Imaging of Lymphatics Background The lymphatics represent the "third circulation" in the body and are an important route of spread for tumors. Lymphangiogenesis is a property of some tumors that allows them to propagate via the lymphatics. This process has been difficult to study because the lymphatics are small and inaccessible. However, by injecting various kinds of macromolecular contrast agents, including radiolabeled, optically labeled and magnetically labeled it is possible to identify the lymphatics and their drainage patterns(1). Moreover, the knowledge gained in this process has implications for the diagnosis and management of primary and secondary lymphedema, which can be a serious complication of cancer therapy. Finally, the development of macromolecular contrast agents has implications for other diseases. Pre-clinical research The MIP is investigating the role of the lymphatics in cancer using imaging methods. We have found that macromolecular agents of between 5-10nm in diameter are ideal for studying the lymphatics based on their rapid uptake and transit to the sentinel nodes. Sentinel node imaging is of importance because current methods rely on cumbersome blue dyes and radioactive sulfur colloid. We have developed a dendrimer that is dual labeled for MRI imaging and optical imaging that would allow the identification of the sentinel node without ionizing radiation using MRI and optical imaging(2). By comparing different sized dendrimers optimization of the lymphatic agent was achieved(3). We found that very small agents leaked from the lymphatics and were unsatisfactory whereas large agents were too slow. Therefore, the Generation 6 dendrimer loaded with Gadolinium and an optical fluorophore appears ideal(3-5). Working with Martin Brechbiel's group we have developed and tested dendrimers with new syntheses that should simplify the chemistry for possible clinical translation(6). We have also used IgG as a carrier molecule for lymphatic imaging and shown it to be a biocompatible and suitably sized for lymphatic imaging. In addition we are exploring the role of quantum dots (QD) in identifying the lymphatics. QDs have the advantages of high quantum efficiency and very narrow emission spectra allowing multiple QDs of different wavelength to be used simultaneously(7, 8). We have employed two color QDs to investigate the drainage of the breast and arm to the axillary lymph nodes and found that some mice had independent drainage systems whereas other mice demonstrated nodes with"watershed" nodes, i.e. nodes that partly drained the breast and partly drained the limb(7, 8). Interestingly the node was divided into separate compartments according to its drainage. The implications of this anatomy for immunologic antigen processing are of potential interest. Additionally we have explored the use of up to 5 QDs of differing wavelength simultaneously in order to establish the complex lymphatic drainage patterns in the head and neck. These studies reveal that it is possible to identify drainage basins for particular regions and show areas of overlapping drainage. We have written a review on the subject of lymphatic imaging over the past year(1). Clinical Translation: We have been collaborating with Boston Children's Hospital to develop a dendrimer-based MR contrast agent for use in human children with primary lymphedema. As of September 07 the material for experimentation in a porcine model has been synthesized by Dr. Brechbiel's group and MIP personnel will assist Boston Children's Hospital in performing these studies. One important spin-off of this work is that we have generated a Gadolinium labeled albumin molecule. Each albumin is labeled with 10-15 Gadolinium atoms. As a consequence this is an excellent imaging agent on MRI and can be used as a surrogate marker. Drs. Ed Oldfield and Russ Lonser have expressed interest in using this agent as a surrogate marker for convection enhanced delivery (CED)agents in patients with brain tumors. Current CED therapy is limited by an inability to monitor the drug. Because Gd-Albumin demonstrates similar convection properties to the agent administered for CED (IL-13 PEG), it will be a useful surrogate marker. The JDC approved the GMP manufacture of Gd-Albumin for use in CED trials to be performed at the Clinical Center. The contract for the GMP production was awarded to the University of Kentucky-Lexington and the GMP synthesis is underway (Sept 07). 1. Barrett, T., Choyke, P. L., and Kobayashi, H. Imaging of the lymphatic system: new horizons. Contrast Media Mol Imaging, 1: 230-245, 2006. 2. Talanov, V. S., Regino, C. A., Kobayashi, H., Bernardo, M., Choyke, P. L., and Brechbiel, M. W. Dendrimer-based nanoprobe for dual modality magnetic resonance and fluorescence imaging. Nano Lett, 6: 1459-1463, 2006. 3. Kobayashi, H., Kawamoto, S., Bernardo, M., Brechbiel, M. W., Knopp, M. V., and Choyke, P. L. Delivery of gadolinium-labeled nanoparticles to the sentinel lymph node: comparison of the sentinel node visualization and estimations of intra-nodal gadolinium concentration by the magnetic resonance imaging. J Control Release, 111: 343-351, 2006. 4. Koyama, Y., Talanov, V. S., Bernardo, M., Hama, Y., Regino, C. A., Brechbiel, M. W., Choyke, P. L., and Kobayashi, H. A dendrimer-based nanosized contrast agent dual-labeled for magnetic resonance and optical fluorescence imaging to localize the sentinel lymph node in mice. J Magn Reson Imaging, 25: 866-871, 2007. 5. Hama, Y., Bernardo, M., Regino, C. A., Koyama, Y., Brechbiel, M. W., Krishna, M. C., Choyke, P. L., and Kobayashi, H. MR lymphangiography using dendrimer-based contrast agents: a comparison at 1.5T and 3.0T. Magn Reson Med, 57: 431-436, 2007. 6. Xu, H., Regino, C. A., Bernardo, M., Koyama, Y., Kobayashi, H., Choyke, P. L., and Brechbiel, M. W. Toward improved syntheses of dendrimer-based magnetic resonance imaging contrast agents: new bifunctional diethylenetriaminepentaacetic acid ligands and nonaqueous conjugation chemistry. J Med Chem, 50: 3185-3193, 2007. 7. Kobayashi, H., Hama, Y., Koyama, Y., Barrett, T., Regino, C. A., Urano, Y., and Choyke, P. L. Simultaneous multicolor imaging of five different lymphatic basins using quantum dots. Nano Lett, 7: 1711-1716, 2007. 8. Hama, Y., Koyama, Y., Urano, Y., Choyke, P. L., and Kobayashi, H. Two-Color Lymphatic Mapping Using Ig-Conjugated Near Infrared Optical Probes. J Invest Dermatol, 2007.